Emerge of NDM-1-Producing Multidrug-Resistant Pseudomonas aeruginosa and Co-Harboring of Carbapenemase Genes in South of Iran

Background: New Delhi metallo-beta-lactamase-1 (NDM-1) is one of the most important emerging antibiotic resistance. Co-harboring three or four carbapenemases is rare and only a few reports exist in the literature. We described the characteristics of the large epidemic outbreaks and reports co-producing blaNDM-1 with the other carbapenemase genes in P. aeruginosa isolates. Methods: This present cross-sectional research was conducted on 369 P. aeruginosa isolates obtained from burn and general hospitals within years 2013 to 2016. Beta-lactamase classes A, B and D genes were identified by PCR method. Modified hodge test (MHT), double-disk potentiation tests (DDPT) and double disk synergy test (DDST) were performed for detection carbapenemase and metallo beta-lactamase (MBL) production of blaNDM-1 positive P. aeruginos isolates. Results: From 236 carbapenem-resistant P. aeruginosa (CRPA), 116 isolates have had MBL genes and twenty-nine isolates were found positive for blaNDM-1. In CRPA isolates, blaIMP-1, blaVIM-2 and blaOXA-10 were identified in 27.5%, 21.1% and 32.2% of isolates respectively, while co-producing blaNDM-1, blaIMP-1, blaOXA-10, co-producing blaNDM-1, blaVIM-2, blaOXA-10 and co-producing blaIMP-1, blaVIM-2 were determined in 11 (4.6%), 8 (3.4%) and 27 (11.4%) of isolates respectively. Conclusion: The finding of this co-existence of multiple carbapenemase resistance genes is threating for public health. Dipicolinic acid is a superior MBL inhibitor in DDPT antique than EDTA in DDST method for the detection of MBL-blaNDM-1 producing P. aeruginosa


Introduction
Pseudomonas aeruginosa is major agents of hospitalacquired pathogens (1). Carbapenemases indicate the most versatile family of beta-lactamase, with a wide spectrum inimitable by other beta-lactam hydrolyzing enzymes (2).
Carbapenems are the last-line treatment of multidrug-resistant P. aeruginosa (MDRP) infections (1,3). Because of the fact that carbapenems are a last resort treatment choice for infections caused by MDRP isolates, the presence of carbapenemresistant strains is becoming a main public health challenge (2,3). Among plasmid-mediated, extended-spectrum beta-lactamases (ESBLs) are commonly known to hydrolyze cephalosporins and metallo beta-lactamases (MBLs) can hydrolyze carbapenems. Resistance to carbapenems can be related to producing carbapenemase enzymes such as serine carbapenemases (containing KPC and GES enzymes) and MBLs (metallo-beta-lactamases) such as imipenemase (IMP), Verona integrin-encoded metallo-β-lactamase (VIM) and New Delhi metallo-β-lactamase (NDM), enzymes and oxacillinases (such as OXA enzymes) (2,4,5). MBLs such as blaVIM and blaIMP are the most clinically important classes of beta-lactamases; but the lately discovered transmissible New Delhi metallo beta-lactamase-1 (NDM-1) is becoming the most menacing in carbapenemase genes (2,6). In addition, most blaNDM-1 strains are resistant to a wide-ranging of other antibiotic groups and transport numerous additional resistance genes for example to aminoglycosides, sulfonamides, macrolides and fluoroquinolones (7). The detection of this co-harboring of multiple carbapenem resistance genes (Simultaneous attendance of both MBL and non-MBL genes) in clinical isolates from supremacy of carbapenems are considered as the last line resort of option for most of the dangerous infections caused by P. aeruginosa, but due to the prevalence of carbapenem-resistant P. aeruginosa (CRPA) isolates these lifesaving antibiotics were compromised in treating the patients with serious sickness (8). The aims study were to identify the carbapenemase classes A, B and D and ESBL determinants among CRPA isolates in burn and non-burn patients. Moreover, identification of blaNDM-1 by three phenotypic methods (include DDPT, DDST and MHT) and comparing with PCR method was evaluated.

Bacterial isolation and identification
During the period from Oct 2013 to Jul 2016, 369 non-duplicate isolates were collected in burns (102 isolates from burn wounds) and general hospitals (267 from various hospital wards). These isolates were collected from teaching hospitals' microbiology laboratories in Ahvaz, Isfahan and Tehran cities from Iran. The isolation and identification of P. aeruginosa were done by the conventional methods and proved by PCR amplification with specific primers for P. aeruginosa gyrB gene with product size 221bp (9).

Phenotypic detection of MBLs
The double-disk potentiation tests (DDPT) and double disk synergy test (DDST) was performed for all blaNDM-1 positive (14,15) for phenotypic detection of blaNDM-1 producing isolates. The bacterial suspension with turbidity equivalent to 0.5 McFarland standard was prepared and cultured on MH agar. Two imipenem and imipenem-EDTA disks and meropenem+Dipicolinic acid (Liofilchem, Roseto degli Abruzzi, Italy) were placed on the surface of the agar at a distance of 4 cm from each other. After 18-24 h of incubation at 35-37 °C, the inhibition zone of imipenem disks with imipenem alone and disks with imipenem plus 750 μg of EDTA were measured. An increase of 7 mm or more in the zone diameter for imipenem-EDTA disk in comparison with imipenem disk alone was considered as a MBLs producing isolate. Moreover, DDPT was interpreted as positive even if a small potentiation inhibition zone was present (14,15).

PCR amplification of resistance genes
DNA of strains was extracted by the DNA extraction set (Sinaclon, Iran) based on the guidelines of the manufacturer. The specific primers were used for different types of carbapenemase (blaNDM, blaIMP, blaVIM, blaKPC, blaGES, blaSPM and blaOXA-10). In this study, pentaplex PCR was used for the rapid detection of MBL genes in CRPA isolates. The pentaplex PCR was optimized successfully to identify the MBL genes. Stepwise optimization of annealing temperature, primer concentration, MgCl2, dNTP and Taq polymerase was performed. The pentaplex PCR gave the excellent results when 5 μL of 10X reaction buffer, 2 μL of 50 mM MgCl2, 1.5 μL of 2.5 mM dNTPs, 0.25 μL of each 10 pmol/μL primer, 0.5 μL Taq polymerase 5 U/μL, 37 μL distilled water and 55 °C annealing temperature were used (Fig.  1). The amplification reactions were carried out in a thermal cycler (Eppendorf AG, Germany), with an initial denaturation 4 min at 94 °C followed by 30 cycles of denaturation 60 sec at 94 °C, annealing 56 °C for blaOXA-10, 59 °C for blaSPM and 55 °C for pentaplex PCR and extension 60 sec at the temperature of 72 °C, with a single final extension of 7 min at 72 °C. The size of PCR products is determined by comparison with a DNA ladder (Sinaclon, Iran) on 1.5% agarose gels stained with ethidium bromide. Sequencing of the amplicons was performed by the Bioneer Company (Bioneer, Daejeon, South Korea). The nucleotide sequences were analyzed using blast in NCBI.  In particular, this collection was included nonduplicate characterized blaVIM, blaIMP and blaNDM-1.  The presence of blaIMP and blaVIM gene were detected in 21.6% (51/116) and 28.8% (68 isolates) of MBL producing isolates, respectively. The full results of antibiotic resistance pattern of blaIMP and blaVIM positive isolates in burns and nonburns isolates showed in Tables 3 and 4. Twenty-four isolates from Ahvaz, 4 isolates from Isfahan and one isolate from Tehran in the collection was found carrying blaNDM-1 and confirmed by sequencing. The prevalence of ESBLs in MBL isolates was 11.2% (13/116) that 3 of them were blaNDM-1 isolates. Nineteen of blaNDM-1 isolates were co-harboring of two genes (blaVIM-2/blaOXA-10 and blaIMP-1/blaOXA-10). Moreover, two blaNDM-1 isolates were co-harboring of three genes (blaVIM-2, blaIMP-1 and blaOXA-10). Moreover, 86.2% (25) blaNDM-1 positive isolates contained blaoxa-10, simultaneously. Furthermore, blaKPC, blaGES and blaSPM genes not found in none of the blaNDM-1 positive isolates. Unexpectedly, the results of DDST and DDPT revealed that 15(51.8%) and 26 (89.7%) of blaNDM-1 positive isolates were MBL producing isolates, respectively.
In the present study, imipenem resistance in burn and non-burn patients was 83.2% and 57.5% respectively. Imipenem was the ninth and fourth effective drugs in burn and non-burn isolates respectively, while in other researches particularly on burned patients in Iran, it was the most effective antipseudomonal antibiotic (24) in 10.8% of 415 isolates In burn patients, ceftazidime (with 26.9% sensitivity( and ertapenem, gentamicin and cefotaxime (with 94.8% resistance( and in nonburn patients amikacin (with 40.5% sensitivity) and imipenem (with 91.8%) resistance were the most and least effective antipseudomonal antibiotics. Even though, amikacin is the most effective antibiotic for infection of CRPA isolates, and also is a good drug for the treatment of non-burn isolates in CRPA isolates, but interestingly, we found that amikacin was a poorly antibiotic for burn infections due to CRPA isolates, the rate of resistance to this antibiotic was 92.4% which is relatively high in burn isolate. Similar to current study, another study among burned patients, reported 97.5% of P. aeruginosa isolates were resistant to imipenem and 90% of isolates resistant to amikacin (25). In Isfahan, surveyed 106 P. aeruginosa was isolated and 62 (58.5%) of isolates were imipenem resistance also MBL detected in 26| (42%) of them (26). In the current study, 21.6% and 28.8% of MBL producing strains, carried blaIMP and blaVIM, respectively. This rate is slightly higher than the result reported in previous studies, which can be a serious concern that may be because of a general increase in the extent of attainment of MBL genes among P. aeruginosa. This genes are found to be located on the class I integron and can hence quickly transfer among P. aeruginosa strains (27,28). Compared to present study, lower resistant to imipenem (n=26, 25.2%), which 19 (73.0%) of them produce MBL, 6 (31.5%) samples had blaVIM gene and 2 (10.5%) had blaIMP gene. Lower percentage of IMP expression (10.5%) than our study has been also reported (29). One general concept has been evidenced that the quick appearance and dissemination of carbapenemase-producing strains is mostly due to the acquisition of blaNDM and blaVIM-2 (7,28). Antimicrobial susceptibility results of VIM and IMP positive isolates in burns and non-burns isolates indicated that high resistance to antibiotics. The corporation of other resistance determinants along with blaVIM confers the phenotype to become resistant to most of the accessible antibiotics (28). Aminoglycosides resistance genes on the similar gene cassette along with blaVIM-2, therefore making the phenotype resistance to gentamicin and amikacin as well (30). Recognition of MBL-producing isolates can be effective for correct treatment of patients especially in burned patients (2). The mortality rate of patients infected with MBL-producing P. aeruginosa was higher (51.2%) than mortality caused by non-MBLproducing strains (32.1%) (31). Aztreonam is not appreciably hydrolyzed by NDM enzymes. Aztreonam was more effective than the carbapenems (31), but our study showed that 62% of these isolates were resistant to aztreonam. This occurrence of blaOXA-10 was inside the range by Golshani (64%), Mirsalehian (74%), but more than other areas; however, blaOXA-10 is prevalent in P. aeruginosa (24,32). Several phenotypic methods to detect MBL production have been developed, comprising the MHT, DDST, DDPT and E-test (15,33). The MHT is the only CLSI recommended carbapenemase-screening method detected the weak carbapenemase activity enzyme. However, PCR is specific for detection of blaNDM. The reports have shown a poor sensitivity of DDST and MHT phenotypic technique for detection blaNDM, furthermore, due to its high false negative results, evaluating the performance of the MBL are needed (15,33,34). In the present study, 51.8% and 89.7% blaNDM-1 isolates were positive in DDST and DDPT methods. There is a need for a more thorough evaluation of blaNDM.in P. aeruginosa (35,36).

Conclusion
These findings imply the importance of blaNDM-1 screening in Iran, which are being reported as potential regions of blaNDM-1 endemicity. The emergence of an acutely drug-resistant strain carrying multiple carbapenemase genes is threating global health. Dipicolinic acid is a superior MBL inhibitor in DDPT than EDTA in DDST method for the detection of MBL-blaNDM-1 producing P.aeruginosa. More research is needed to detect the blaNDM-1 source.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.